Advances in genetic engineering techniques have allowed numerous physiologically active polypeptides and proteins to be provided in a stable manner by cell culturing methods, for application in the treatment or prevention of diseases. Such polypeptides, however, generally have a short half-life in vivo due to their extremely rapid enzymolysis, metabolism and the like, and in most cases it has not been possible to obtain satisfactory effects when they are administered as drugs. A great deal of research has been conducted to date toward solving this issue, with focus on modification of the polypeptides and proteins with polymers or their sustained-release formulations.
For example, polyethylene glycolation is a polymer modification technique currently used for clinical purposes. Extension of in vivo half-life has been achieved for interferon and the like, thus allowing some degree of sustained effect. This has resulted in less frequent administration and thus reduced burden on patients, but such polymer-modified proteins generally exhibit lower activity due to the modification, and it has been difficult to control the modification sites and modification rates in a reproducible manner.
Microcapsules are also currently used in the clinic as a sustained-release technology. This technology is implemented by employing in vivo-degradable polylactic acid or polylactic acid/glycolic acid copolymer as the base for inclusion of a drug into fine particles. However, the particle size is usually in the micrometer range and is not suitable for intravenous administration. Microcapsules with particle sizes reduced to nanosize have been reported, which are subjected to surface modification to control their uptake into the reticuloendothelial system of the liver or spleen following intravenous administration (Adv. Drug Deliv. Rev. 17, 31-48 (1995)). However, the particle sizes obtained by such methods are at minimum a few hundred nanometers (Int. J. Pharm. 149, 43-49 (1997)), while the surface modification is laborious and it has also been difficult to control the organ distribution in a reproducible manner.
Liposomes using phospholipids may also be mentioned as examples of sustained-release technology currently used in the clinic (Pharm. Tech. Japan 19, 99-110 (2003)). The advantage of liposomes is their low toxicity and antigenicity, because phospholipids are biological substances, and the fact that altering the lipid composition allows encapsulation of numerous bioactive substances such as water-soluble drugs, fat-soluble drugs, macromolecules, proteins, nucleic acids and the like. However, such liposomes do not necessarily have adequate drug retention properties. Specifically, the amounts of drugs that can be encapsulated per unit liposome formulation are currently inadequate and more efficient methods are desired. In addition, the problems such as insufficient stability in vivo and difficulty of industrial production have still not been satisfactorily overcome.
Polymer micelles may be mentioned as a sustained-release technology that is currently being investigated in the clinic as a means of solving these problems (Br. J. Cancer 93, 678-697 (2005), Br. J. Cancer 92, 1240-1246 (2005)). Polymer micelles can be produced using block copolymers composed of hydrophilic polymers and hydrophobic polymers. In water, these block copolymers generally form polymeric micelles with the core comprising of hydrophobic segments, and therefore exhibit excellent properties in terms of fat-soluble drug encapsulation, solubilization and sustained release. (Japanese Patent Publication No. 2777530).
Such polymer micelles are also studied for encapsulation and sustained release of water-soluble drugs. For example, one method of encapsulating adriamycin as a water-soluble compound into polymer micelles involves chemical linkage of the drug to the side chains of the hydrophobic polymer (Japanese Patent Publication No. 2694923). Other alternative methods have also been disclosed for efficient encapsulation by introducing electrostatic interaction between polymer micelles and a peptide, such as a method in which negatively charged functional groups are introduced into the side chains of hydrophobic segments in a block copolymer, for drugs with chargeable substances such as a positively charged basic peptides (Japanese Patent Publication No. 2690276), or a method in which a biodegradable polymer with a carboxyl group, such as polylactic acid or poly(lactic-co-glycolic acid), is added (WO2005/023230). However, these cannot be applied for water-soluble drugs with large molecular weights, and especially proteins and polypeptides. Japanese Patent Publication No. 2690276 discloses examples of encapsulating proteins into micelles. However, the micelles themselves are poorly stable and, when actually administered to the body, are believed to undergo an immediate breakdown, because they have no hydrophobic portions and form only under electrical charge.
A method for stabilizing micelles encapsulating polyelectrolytes has been disclosed, wherein polyion complex micelles with a core-shell structure, formed of a polyelectrolyte and a block copolymer containing hydrophilic and electrically charged segments, have at least one thiol group carried on the electrically charged segments forming the core so that stability is enhanced by crosslinking with disulfide bonds between the electrically charged segments, via the thiol groups they carry (Japanese unexamined Patent Publication (Kokai) No. 2001-146556). During actual use, however, after administration by intravenous injection, the micelles dissociate due to dilution or interaction with serum proteins or undergo interaction with proteins having SS bonds in the molecules. These interactions lead to inactivation of the proteins and destabilization of the is micelles. Therefore, this method cannot be applied for most proteins or polypeptides.
In order to increase the therapeutic effects of physiologically active polypeptides and proteins it is necessary to provide polymer micelles that stably and efficiently encapsulate the physiologically active polypeptides and proteins while allowing their release in a controlled manner, as explained above, but at the current time no such micelles exist that elicit a low immune response and that can be applied to a wide range of physiologically active polypeptides and proteins.
The following techniques have also been proposed to date in an attempt to fulfill the specifications mentioned above, in order to increase the therapeutic effects of physiologically active polypeptides and proteins, but not all of them have been successful.
(A) Japanese Unexamined Patent Publication (Kohyo) No. 2004-525939 relates to a colloidal suspension of nanoparticles, based on polyamino acid blocks and polyalkylene glycol-type hydrophilic polymer blocks, such as polyethylene glycol (PEG). Since formation of drug (protein or polypeptide) nanoparticles is based on adsorption of the drug onto nanoparticles, the protein or polypeptide is present on the nanoparticle surfaces. Specifically, it is believed that attack by digestive enzymes in the body causes rather rapid decomposition of the protein or other substance on the nanoparticle surfaces, resulting in its inactivation. In addition, since the isoelectric points of the proteins and polypeptides that are to be encapsulated are not considered in forming the nanoparticles, the release may be relatively rapid, making it impossible to obtain a long-lasting effect.
(B) European. Patent Publication No. EP1084172B1 relates to delivery of nucleic acids, in particular, using palmitoyl poly-L-lysine polyethylene glycol or palmitoyl poly-L-ornithine polyethylene glycol, in the presence of cholesterol. The particle sizes of the fine particles obtained by this technique are a few hundred nanometers at the smallest, and since they rapidly accumulate in the reticuloendothelial system after intravenous administration, they cannot easily produce long-lasting effects.
(C) Japanese Unexamined Patent Publication (Kokai) No. 11-269097 relates to fine particles with functions such as organ directivity and sustained release, of which the base is a block copolymer comprising a biodegradable polymer as hydrophobic segments and polyamino acid as hydrophilic segments. This strategy is characterized by using biodegradable polyamino acid as the hydrophilic segments, but compared to polyethylene glycol, it is expected to have higher immunogenicity and increased interaction with serum proteins after intravenous administration, leading to shorter retention in blood circulation, making it impossible to obtain a long-lasting effect.
(D) U.S. Pat. No. 6,090,925 discloses a method in which an acetate or phosphate buffering solution containing polyethylene glycol and polyvinylpyrrolidone is added to an aqueous solution of a low molecular compound or peptide which is to be encapsulated, and then a polymer such as serum albumin having an isoelectric point near the pH of the buffering solution is added thereto and microparticles are formed by heating and cooling steps. Because this method includes a heating step at about 70° C., it is considered poorly suitable for heat labile proteins.